Acanthamoeba myosin II rod is a long alpha-helical dimeric coiled-coil with a flexible hinge containing a helix-breaking proline. The thermal stability of the complete rod domain of myosin II (residues 849-1509), a mutant in which the hinge proline was replaced with alanine (P398A), and a mutant with the whole hinge region deleted (delta 384-408) in 0.6 and 2.2 M KCl, pH 7.5 was determined. In addition, analytical ultracentrifugation studies showed that the myosin II rods sedimented as monodisperse dimers with sedimentation coefficients s(20,w) = 3.8 S (wild-type) and 3.6 S (P398A and delta 384- 408). Thus, the mutant rod proteins had lower s(20,w) values and higher frictional ratios than those of the wild-type rod, which is consistent with the hinge mutants having a reduced flexibility. Circular dichroism (CD), differential scanning calorimetry (DSC) and HPLC gel filtration showed that the thermal unfolding of the myosin II rod is reversible, highly cooperative and coupled to a dissociation of the two chains. The CD and DSC data are consistent with a two-state mechanism (Tm = 40 degreesC, deltaH = 400 kcal/mol) in which the dimeric rod unfolds with the concomitant formation of two unfolded monomers. We found no evidence for independent unfolding of the two rod domains that are separated by the hinge region. The only difference observed in the unfolding of the mutant rods from that of the wild-type was an approximate 2 degreesC increase in the thermal stability of the hinge-deletion mutant. Thus, the mechanism of unfolding of Acanthamoeba myosin II rod is different from non-two-state transitions observed for skeletal muscle myosin rod and tropomyosin. The cooperative unfolding of the entire coiled-coil rod of myosin II may underlie the previously reported regulatory coupling between N-terminal heads and the C-terminal tail. [Biochemistry 36, 7876-7883, 1997.]